The invention relates to a microoptical sensor for detecting chemical substances, this sensor having a planar optical waveguide which comprises a solid substrate having layered on its top surface a waveguiding layer and a diffraction grating, situated on or in the immediate vicinity of the waveguiding layer, for coupling and decoupling coherent light applied thereto.
The invention further relates to a microoptical method for detecting chemical substances with a microoptical sensor of the kind indicated above, which method comprises contacting the surface of the planar optical waveguide with a sample containing the substance to be detected, coupling coherent light into said waveguiding layer, which light propagates therein as a guided light wave, then decoupling said light wave out of the waveguiding layer.
Chemical changes, e.g. molecular additions, at or in the immediate vicinity of the waveguiding layer are detected, for example, by measuring the relative intensity of the decoupled beams of light. An alternative detection method comprises simultaneously coupling into the waveguide two coherent (e.g. orthogonally polarized) beams of light and measuring the relative phase (phase difference) of two decoupled beams by interference of said sub-beams, which are generated by the two jointly excited (orthogonally polarized) modes in the waveguide.
Many of the conventional test methods used in biomedical diagnosis are based on the use of solid carrier substrates, e.g. balls or pellets, coated with a chemo-sensitive molecular coating. For analysis, the patient's sample, e.g. serum or plasma, is brought into contact with the carrier substrate. Molecules, e.g. biomolecules, dissolved in the sample, and required to be detected, enter into a specific bond with the chemo-sensitive coating. Typically, the chemo-sensitive coating consists of biomolecular recognition elements, such as antibodies, receptors, enzymes or DNA-strands.
In conventional tests, detection of the molecules bound to the chemosensitive coating is usually effected indirectly by means of a second dissolved reaction partner labelled with a radio isotope, a fluorophore or an enzyme. The labelled molecules have the specific property of coupling to those binding sites which have been left unoccupied on the chemo-sensitive coating, or of coupling to the free end of the molecules to be detected, which in turn bind to the chemo-sensitive coating, the latter is called "sandwich" technique. The resulting concentration of the labelled molecules on the carrier substrate is determined by a suitable measurement technique. The concentration of the substance to be detected in the sample is concluded therefrom.
Planar optical waveguides consist of a thin dielectric layer or coating on a transparent carrier substrate. (For a tutorial introduction see, for example T. Tamir, Integrated Optics, Springer, Berlin 1985). The medium covering the waveguiding layer is known as the superstrate and may, for example, be gaseous or liquid. Light, e.g. a laser beam, coupled into the waveguide is guided by total internal reflection in the waveguiding layer, provided the substrate and the superstrate have a lower refractive index than the dielectric layer therebetween. The propagation of the optical wave in the waveguide is restricted to a number of discrete modes. The phase velocity of the guided light wave is c/N, where c is the velocity of light in vacuum and N is the effective refractive index of the excited waveguide mode. The effective refractive index N depends on the optical parameters of the waveguiding structure, i.e. on the thickness and refractive index of the thin waveguiding layer and on the refractive indices of the substrate and superstrate material. The transverse field distribution of the modes rapidly falls off outside the waveguiding layer. The effective thickness d.sub.eff of the waveguide is defined as the sum of the geometric thickness d of the waveguiding layer and the penetration depths of the evanescent fields into the substrate and the superstrate. By the use of suitable high-refractive materials for the waveguiding layer, waveguides with a d.sub.eff of less than one wavelength of the guided light can be realized. Under these conditions the penetration depth of the evanescent fields into the substrate and superstrate is only a fraction of the wavelength.
The field of the guided mode, which is highly spatially confined at the substrate surface, is ideally suited for sensing chemical changes taking place at or in the immediate vicinity of the waveguiding layer. Optical measurement techniques based on a waveguiding structure, also known as "integrated-optical" techniques, are increasingly gaining significance for surface analysis and optical sensor systems. Sensing schemes are known in which changes of the propagation constant (effective refractive index) of the guided modes, and/or changes in light intensity caused by absorption of the guided modes are utilized to detect (chemical) changes at the interface between the waveguiding layer and the superstrate and/or in the volume of the waveguiding layer.
Selective detection of specific substances in the sample covering the waveguide is achieved by an additional chemo-sensitive layer on the wave-guiding layer. Such additional layer is capable of binding selectively the molecules to be detected. This results for example, in a change of the effective refractive index of the guided mode. The interaction of the guided mode with the sample takes place via the evanescent field, whose penetration depth into the superstrate is typically greater than the thickness of the additional chemo-sensitive layer.
According to the known prior art, the light is coupled into the waveguide by focusing a laser beam on its end face (butt-face coupling), or by means of a diffraction grating (grating coupling), in the latter case the beam to be coupled is incident on the waveguiding layer from the side of the substrate or superstrate. Butt-face coupling makes great demands upon the mechanical positioning of the coupling lens, particularly for extremely thin surface-sensitive waveguides with a d.sub.eff of less than 1 micrometer. With grating couplers it is possible easily to couple a laser beam into and decouple it from a waveguide without the use of focusing optics.
Arrangements are known in which laterally bounded grating structures on the waveguiding layer are used for coupling an incident beam into the waveguides or decoupling an excited waveguide mode. A laser beam is coupled in if it meets the region of the waveguide provided with the grating structure at a specific angle of incidence dependent upon the grating period and the effective refractive index. The excited mode passes, for example, through a portion of the waveguide situated between two spatially separated grating regions and is decoupled on meeting the second grating region.
By measuring the intensity of the decoupled guided mode, the absorption by molecules situated at the surface of the waveguide can be detected with high sensitivity. Coupling of the incident wave to the guided wave comes into effect only within the area of the grating region. Coupling and decoupling via the grating has the character of a resonance. The resonance angle for optimal coupling is dependent upon the grating period and on the effective refractive index of the guided mode. A change of the effective refractive index, due e.g. to addition of molecules at the surface of the waveguide, results in a shift of the angle of resonance at which the laser beam is coupled in or decoupled. By measuring the angle of incidence at which the guided wave can be excited, a molecular surface coverage in the grating region of the waveguiding layer can be detected with submonomolecular sensitivity (cf. K. Tiefenthaler and W. Lukosz, "Integrated optical switches and gas sensors", Optics Letters vol. 9, No. 4, 1984, pp. 137-139, and K. Tiefenthaler and W. Lukosz, U.S. Pat. No. 4,815,843, 1989).
Another well-known technique for detecting adsorbate layers is based on the optical excitation of surface plasmons at the surface of a thin metal layer with or without the use of a diffraction grating. The coherent excitation of the free electrons of a metal in the form of a surface wave propagating along the surface of the metal is known as surface plasmon. The electromagnetic field of the surface plasmon is spatially confined at the metal surface. The transverse field distribution has a maximum at the surface and falls off exponentially in the metal and in the superstrate. The plasmon wave is damped by ohmic losses in the metal. The propagation distance of the surface plasmon is, for example, 22 micrometers for pure silver at a wavelength of 514 nm. If molecules adsorb at the metal surface, the propagation constant (phase velocity) of the surface plasmon propagating along the interface changes.
The sensitivity with which this change of the propagation constant can be measured is limited by the relatively short propagation distance of the surface plasmon. Various optical configurations are known which utilize the resonant excitation of surface plasmons in order to detect molecular adsorbate layers on metal surfaces (cf. see B. Liedberg, C. Nylander, and I. Luridstrom, "Surface plasmon resonance for gas detection and biosensing", Sensors and Actuators 4, 1983, pp. 299-304 and EP 0 112 721).
A universal method of characterising thin layers on planar surfaces is ellipsometry, which is based on the measurement of the state of polarization of light reflected at the surface. A light beam impinging on the surface at a certain angle of incidence experiences on reflection a change in the relative amplitude and phase of the electromagnetic field components polarized parallel and perpendicular to the plane of incidence. The incident beam of light is preferably circularly or linearly polarized. The state of polarization of the generally elliptically polarized reflected beam is analysed. This information is used to determine the thickness and the refractive index of the thin layer (cf. R. Azzam et al., Physics in Medicine and Biology 22, 1977, 422-430, P. A. Cuypers et al., Analytical Biochemistry 84, 1978, 56-57).
Upon a single reflection of the beam at the surface for analysis, the changes in the state of polarization due to a molecular adsorbate layer is very small. This can be explained by the fact that the interaction of the incident beam with the adsorbate layer is restricted to a distance of the order of the layer thickness. Minor changes in the state of polarization after the passage of the incident and reflected beam through the substrate carrying the adsorbate layer to be detected, or after the passage of a cell attached to the substrate and containing the sample fluid, restrict the accuracy of an ellipsometric measurement.